Enhancers predominantly regulate gene expression during differentiation via transcription initiation

Gene transcription occurs via a cycle of linked events, including initiation, promoter-proximal pausing, and elongation of RNA polymerase II (Pol II). A key question is how transcriptional enhancers influence these events to control gene expression. Here, we present an approach that evaluates the level and change in promoter-proximal transcription (initiation and pausing) in the context of differential gene expression, genome-wide. This combinatorial approach shows that in primary cells, control of gene expression during differentiation is achieved predominantly via changes in transcription initiation rather than via release of Pol II pausing. Using genetically engineered mouse models, deleted for functionally validated enhancers of the α- and β-globin loci, we confirm that these elements regulate Pol II recruitment and/or initiation to modulate gene expression. Together, our data show that gene expression during differentiation is regulated predominantly at the level of initiation and that enhancers are key effectors of this process. Larke et al. use a modification of Start-seq (scaRNA-seq) to simultaneously quantify initiation and pausing. Combined with nascent transcription assays, they show that initiation is the predominant point of transcriptional control during differentiation. Specific enhancer deletions cause loss of Pol II recruitment or initiation rather than affecting Pol II pausing.